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REVIEW

CHILEAN J. AGRIC. RES. - VOL. 69 - N 2 - 2009

USE OF ENZYMATIC BIOSENSORS AS QUALITY INDICES: A SYNOPSIS

OF PRESENT AND FUTURE TRENDS IN THE FOOD INDUSTRY
Liliana Serna Cock1*, Ana Mara Zetty Arenas1, and Alfredo Ayala Aponte2

ABSTRACT
Biosensors are an important alternative in the food industry to ensure the quality and safety of products and process
controls with effective, fast and economical methods. Their technology is based on a specific biological recognition
element in combination with a transducer for signal processing. The use of enzymatic biosensor technology in food
processing, quality control and on-line processes is promising compared to conventional analytical techniques, as
it offers great advantages due to size, cost, specificity, fast response, precision and sensitivity. This article reviews
the development and use of some enzyme biosensors in the food industry, describes the most important application
areas and analyzes the current situation and future possibilities. In conclusion, enzymatic biosensors are a tool with
broad application in the development of quality systems, risk analysis and critical control points, and the extent of
their use in the food industry is still largely limited by the short lifetime of biosensors, in response to which the use
of thermophilic enzymes has been proposed.
Key words: biosensors, enzymes, analysis in food, safety, quality, control processes.

INTRODUCTION
In recent decades increased knowledge about the
biological capacity of enzymes has made it possible to
create a new generation of products and processes. Among
these products are notably biosensors, which represent a
powerful alternative to conventional analytical technique
(Velasco-Garca and Mottram, 2003). This technology
has advanced considerably in recent years, basically
because of the creation of devices applied in the area of
biomedicine. These advanced technologies have been
gradually transferred horizontally to other sectors, such as
the environment and the agro-food industry.
A biosensor is defined as a compact device for analysis
that incorporates a biological or biomimetic recognition
element (nucleic acid, enzyme, anti-body, receptor, tissue,
cell) associated with a transduction system that allows for
processing the signal produced by the interaction between
the recognition element and the analyte. The principle of
detection of a biosensor is based on the specific interaction
between the analyte of interest and the recognition element.
As a result of this specific interaction, changes are produced
Universidad Nacional de Colombia sede Palmira, Facultad de
Ingeniera y Administracin, Carrera 32 Chapinero, Va Candelaria,
Palmira, Colombia.
*
Corresponding author (lsernac@palmira.unal.edu.co).
2
Universidad del Valle, Facultad de Ingeniera, Edificio 338, Espacio
2016, Ciudad Universitaria, Cali, Colombia.
Received: 11 December 2007.
Accepted: 05 May 2008.
1

in one or several physical-chemical properties (pH, electron

transference, heat transference, change of potential or mass,
variation of optical properties, etc.). These changes are
detected and can be measured by a transductor (VelascoGarca and Mottram, 2003). This system transforms the
response of the recognition element into an electronic
signal indicative of the presence of the analyte under study
or proportional to its concentration in the sample.
Biosensors currently represent powerful tools for
analysis with numerous applications in the agro-food
industry, mainly in biotechnological instruments (Mello
and Kubota, 2002). The most important characteristics of
these devices to be competitive with other technologies in
the agro-food industry are their specificity, high sensitivity,
short response time, their capacity to be incorporated into
integrated systems, the facility to automate them, their
capacity to work in real time, their versatility and low
production cost (Rasooly, 2001; Mello and Kubota, 2002;
Velasco-Garca and Mottram, 2003).
In recent years, the number of scientific investigations
and reviews on biosensors has been very high, which reflects
the considerable interest in the theme. Ironically, there is a
lag between the high level of scientific and technological
development and the limited use of these devices in the
agro-food sector (Velasco-Garca and Mottram, 2003)
basically because of structural characteristics of the sector,
such as legislation, methodological inertia, absorption
capacity and environmental factors. The development
of the diverse technologies involved in the design and
construction of biosensors has allowed in recent years for

CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 69(2):270-280 (APRIL-JUNE 2009)

L. SERNA et al. - USE OF ENZYMATIC BIOSENSORS AS QUALITY INDICES

resolving technical difficulties and personalized design of

biosensors that, from the technical point of view, cover
practically all needs.
The development of biosensors is described in
numerous works, the majority in the areas of clinical,
environmental, agricultural and biotechnological
applications (Tothill, 2001). Their use in the food sector
is convenient to ensure the quality and safety of foods
(Luong et al., 1991). The potential uses of biosensors
in agriculture and food transformation are numerous
and each application has its own requirements in terms
of the concentration of analyte to be measured, required
output precision, the necessary volume of the sample,
time required for the analysis, time required to prepare
the biosensor or to reuse it and cleanliness requirements
of the system (Velasco-Garca and Mottram, 2003).
Biosensors have been adapted to detect or measure
analytes in on-line systems, that is, simultaneous with
food processing (Rasooly, 2001). Hazard Analysis and
Critical Control Point (HACCP) is generally regarded
as the most effective system to ensure food safety. It is
highly useful in verifying that processes are under control.
The high sensitivity of enzymatic biosensors allows for
the detection of microorganisms such as Escherichia
coli, Salmonella sp., Staphylococcus aureus, pesticides
and herbicides, among others, in hours and/or minutes
(Fitzpatrick et al., 2000; Killard and Smyth, 2000).
Innovation and development in the food industry are
guided by the central principles: food safety and quality
(Mello and Kubota, 2002; Ferreira et al., 2003). The
increasing complexity of the food chain requires, among
other things, the development of effective traceability
systems that guarantee the solidity of all the links. In both
cases, the priority is to develop and install control systems
such as biosensors that involve molecular methods of
detection, analysis and diagnosis that are rapid, highly
sensitive and automated tracing for a wide range of agents
that threaten food safety.
Given the applicability of biosensors, this article reviews
the development and use of some enzymatic biosensors in the
food industry, describing the three main areas of application:
food safety, food quality and process control, the current
situation and future possibilities. As well, there is a brief
commentary on the aspects of catalytic biosensors and their
classification according to the type of interaction established
between the recognition element and the analyte.
Enzymatic biosensors
A catalytic biosensor can be described as a compact
analytic device that incorporates a biological sensing
element, closely connected to or integrated with a
transductor system (Velasco-Garca and Mottram, 2003).
Biological sensors include enzymes or multi-enzymatic

271

mediums, cellular organelles, complete cells or animal

or vegetal tissue (Davis et al., 1995; Mello and Kubota,
2002; Wilson and Gifford, 2005), which are used to detect
the presence of any of the substrates that participate in
the reaction by detecting the disappearance of a known
substrate distinct from the substrate being sought, or by
the appearance of a known product; biological sensors are
not consumed and can be reused (Gajovic et al., 2000;
Wilson and Gifford, 2005).
In enzymatic biosensors a reaction occurs catalyzed by
an enzyme in which the union of the substrate is produced
in a concrete region of the enzyme termed the active
center, which upon forming the products is recovered
and can begin a new reaction cycle. The use of enzymes
as biological recognition elements was very popular
in the first generation of development of biosensors
owing to their commercial availability or the facility for
isolation and purification of diverse sources (Luong et
al., 2008). Subsequently, other advantages were found in
using enzymes in recognition biosensors, such as rapid
response, high selectivity, the possibility of regeneration
and the simplicity involved in constructing the devices
(Hall, 2002). In numerous cases multi-enzymatic chains
are employed, where the enzyme that generally recognizes
the analyte does not act directly on it, but rather interacts
with some product derived from it. This technique is often
used, for example, with some sugars, where enzymes that
are used react with the products of hydrolysis of the same.
Among the enzymes that are commercially available, the
most often used in biosensors are oxidoreductase, notably
among which are glucose oxidase, horseradish peroxidase
and alkaline phosphatase (Rogers and Mascini, 1998;
Laschi et al., 2000), because they are very stable catalyzing
reactions of oxide reduction (Mello and Kubota, 2002).
More than 2000 articles on enzyme-based biosensors
were found in the literature and this is because of the
need to examine blood glucose (Tothill, 2001; DOrazio,
2003) and the feasibility of construction of the same. In
the majority of the applications, the detection limits are
satisfactory or excessive, but the stability of the enzymes
and the capacity to maintain enzymatic activity over a
long period of time continues to be problematic, which is
generally resolved by immobilizing the enzymes (Tothill,
2001; DOrazio, 2003). Immobilized enzymes offer
advantages for application in different types of industrial
processes and are adaptable to new engineering designs
(Krajewska, 2004), for which it is important to increase the
affinity of the enzyme to the substrate, reduce inhibition,
increase the optimal pH interval and reduce possible
microbial contamination (Arroyo, 1998). Basically, in
enzymatic biosensors the enzymes are immobilized in a
potentiometric, amperometric, optometric, calorimetric or
piezoelectric transductor (Davis et al., 1995).

272

In some cases electro-active interference caused

by endogenous compounds in the sample for analysis
becomes significant and needs to be eliminated.
Currently, glucose oxidase continues to be the most stable
and specific enzyme that can be easily obtained in large
quantities (Luong et al., 2008).
On the other hand, there are enzymes that cannot be
used because they are not sufficiently stable or because
their purification is difficult or too costly, because of which
cells from bacteria, fungus, protozoa and higher organisms
are being used (Mello and Kubota, 2002). Instead of being
purified, these cells are used as biological recognition
elements, taking advantage of their diverse multi-cellular
enzymatic systems that possess the capacity to metabolize
different organic compounds, generating distinct products
such as ammonia, carbon dioxide, acids, sugars, vitamins
and nitrogenated compounds, among others, which can
be detected in the biosensors, and in turn can be used to
detect compounds that inhibit microbial respiration as
toxic and contaminating substances (DSouza, 2001). As
well, the cells offer the facility to be modified genetically
to improve their activity or to produce specific enzymes
that do not normally appear.
One of the most important limitations in the use of
complete cells is the diffusion of substrates and products
by means of the cellular membrane, with which a slower
response is obtained in comparison to purified enzyme
biosensors, and with less specificity owing to reactions
catalyzed by other enzymes present in the cell (DSouza,
2001). These limitations can be reduced through the
permeabilization of the cellular membrane by means of
enzymatic processes with lysozyme, papain, chemical
processes with detergents and physical processes with
freezing and thawing (Mello and Kubota, 2002). These
processes permit an increase in porosity of the membrane,
allowing for better incorporation of the analyte and
impede the escape of the intercellular macromolecular
compounds, while the enzymes at the same time allow the
co-factors that intervene in the catalytic reactions to leave
to the exterior of the cell. Consequently, such treatments
provoke cellular inviability, which limits their use to
applications that do not require cellular regeneration
of co-factors or metabolic respiration, as is the case of
glucose oxidase, -galactosidase, amino acid oxidase and
invertase (DSouza, 2001).
Cells can be immobilized in membranes of cellulose
acetate, or be trapped in a matrix, such as agar gel
(Patel, 2002; Tatsumi et al., 2006; Setti et al., 2007) in
a simpler and more economical way than enzymatic
immobilization.
Other systems of catalytic biosensors instead of
using complete cells or isolated multi-enzymatic include
subcellular organelles or tissue, that contain more specific

CHILEAN J. AGRIC. RES. - VOL. 69 - N 2 - 2009

complete enzymatic systems, as is the case of thylakoids,

complete chloroplasts or mitochondria, which are doublemembrane organelles that have enzymatic systems related
to obtaining energy. Such organelles are used in the
detection of toxic agents such as pesticides, heavy metals
or detergents that can inhibit enzymatic systems (DSouza,
2001). In the same manner, there are determined tissues that
according to their physiological function in the organism
produce specific enzymes or enzymatic systems, such as
leaves, roots, fruits or seeds, in sliced or in homogenized
form; such tissue are often associated with electrochemical transductors (Li et al., 2002; Wilson and Gifford,
2005; Mei et al., 2007). For example, for the detection
of phenol impregnated in salmon, salmon tissue is used
in an electrochemical biosensor of carbon and tyrosinase
mixed with electropolymerized pyrrole (Tingry et al.,
2006); slices of potato are used for the determination of
mono and polyphenoles (Solanum tuberosum L.) because
of their high content of polyphenol oxidase enzymes,
together with oxygen electrodes (DSouza, 2001; Kulys
and Vidziunaite, 2003); for the determination of alcohol,
homogenized fungus Agaricus bisporus is used (Akyilmaz
and Dinckaya, 2000; Kulys and Vidziunaite, 2003); for the
determination of diamines like putrescine and cadaverine,
tissue is used from chemiluminescent plants, based on the
enzymatic conversion that takes place in the column of
tissue of the plants to produce hydrogen peroxide (Mei et
al., 2007), among many others examples.
The application of more than one sensor channel for
one or more species in a unit using enzyme-electrode
type amperometric sensors has led to the development of
several multi-sensors based on principles of amperometric
detection (Silber et al., 1994; Miertus et al., 1998). The use
of electro-chemical transductors of various channels allows
for the construction of biosensors that can simultaneously
analyze three or more species and in this manner optimize
selectiveness and reliability in comparison to sensors with
only one substrate (Glazier et al., 1988).
Potential applications of enzymatic biosensors in the
agro-food industry
Applications in food safety. The concept of food
safety involves ensuring the production and marketing
of harmless food, with this, ensure the health of the
consumer. The quantity and types of food additives
incorporated into food products are regulated by
the legislation of each country, the detection and
quantification of which are important to prevent fraud
and malpractice by manufacturers, allergies and other
adverse effects to determined groups of the population
(Zinedine et al., 2007). Because of this, special attention
has been given to studying the way to detect the presence
of contaminants, such as residues of pesticides, fertilizers,

L. SERNA et al. - USE OF ENZYMATIC BIOSENSORS AS QUALITY INDICES

heavy metals and organic compounds, given that the

majority of these have a high level of toxicity. Based
on this need biosensors are used to detect xenobiotic
substances, substances external to the food product such
as additives and pesticides and components of the food
itself like toxines of diverse origins (Xavier et al, 2000;
Patel, 2002). The traditional methods to identify food
contaminants include physiochemical, serological and
biological tests, however many of these require large
quantities of prepared samples, analysis time and lack
sufficient sensitivity and selectivity.
The development of catalytic biosensors in food
additive analysis generally employs enzymes as
recognition systems. This development is described in
several investigations, among these notably are analysis
of aspartame with carboxyl esterase, alcohol oxidase,
caboxypeptidase, L-aspartase, peptidase, aspartate
aminotransferase, glutamate oxidase and -chymotrypsin
(Odaci et al., 2004); analysis of sorbitol with sorbitol
dehydrogenase and nicotinamide adenine dinucleotide
(NAD+) (Saidman et al., 2000); analysis of benzoic
acid with tyrosine (Morales et al., 2002) and analysis
of sulphites with sulphite oxidase; all developed with
a system of amperometric transduction. In some cases
interference reactions have been observed that reduce
the efficacy of these devices, as occurs with biosensors
used to detect sorbitol which also interact with another
artificial edulcorant, xylitol; and with devices designed
for the determination of benzoic acid by the presence of
other antioxidants such as butyl hydroxyanisole (BHA)
and propyl gallate (Patel, 2002). Table 1 presents the
main biosensors used in the detection of these types of
compounds in food and water; among these are devices
based on the inhibition of enzymatic activities that
incorporate enzymes such as cholinesterases (acetyl
and/or butyrylcholinesterases), tyrosinase or alkaline
phosphatase; and units in which reactions are catalyzed
that affect the analyte of interest, which include hydrolases,
reductases, etc. (Nunes et al., 1998; Bachmann et al.,
2000; Panfili et al., 2000).
The different pesticides used in food production
can accumulate in the fatty tissue of animals, while the
excessive use of fertilizers contaminates ground water
with nitrates, nitrites and phosphates (Cosnier et al., 1998;
Moretto et al., 1998). For the detection of herbicides such
as phenyl urea and triazines, which inhibit photosynthesis,
biosensors have been designed with membrane receptors
of thylakoid and chloroplasts, photosystems and reaction
centers; or complete cells such unicellular alga and
fenilureas and triazines, for which mainly amperometric
and optical transductors have been employed (Patel,
2002).

273

There are also other substances potentially toxic for

humans with a major impact on the environment that can
reach the food chain accidentally, such as contaminating
residues present in water and soil, among these byproducts from diverse industrial processes (dioxins), used
as dielectric or hydraulic fluid agents (polychlorinated
biphenyls or PCBs) or generated in the burning of fossil
fuels or wood (polycyclic aromatic hydrocarbons or PAHs),
benzene, toluene and xylene (named BETX) and derived
phenolics; immunosensors, enzymatic biosensors and
biosensors with complete cells are used for the detection
of these organic compounds (Hedenmo et al., 1997; Patel,
2002). Likewise, devices have been designed to determine
the levels of heavy metals such as arsenic, cadmium,
mercury, lead, among others, in samples of water and soil,
which incorporate genetically modified microorganisms
and enzymes such as urease, cholinesterase, glucose
oxidase, alkaline phosphatase, ascorbate oxidase and
peroxidase (Tsai et al., 2003), the transduction systems in
these devices are notably electrochemical and optical, as
indicated in Table 1.
On the other hand, foods can naturally present antinutritional compounds that can generate disorders in
the consumer, given that they hinder absorption and
metabolize distinct nutrients causing a deficiency of the
same. Table 2 presents some examples of biosensors used
in the detection of anti-nutrients.
Applications in food quality. The term food quality is
related to nutritional value, acceptability and safety. The
latter was analyzed in the previous section and the others
are evaluated in function of parameters such as freshness,
appearance, flavor, texture and chemical (Vadiuambal
and Jayas, 2007). The composition of the foods allows
for characterization and verification if the food contains
elements to enrich the food such as vitamins and/or
minerals. To evaluate food composition distinct biosensors
have been developed, which are described in Table 3.
Various food labeling regulations recognize the
importance of determining freshness, establishing
guidelines for the use of the term fresh in relation to
food (FSA, 2004). One way to determine freshness is
through evaluation of the composition of products such
as meats, fish, fruits and vegetables, given that during
periods of storage compounds can by synthesized that
produce abnormal odors and flavors and are prejudicial
to the health of the consumer. Table 4 lists the biosensors
developed to evaluate the freshness and useful life of
foods.
Some of the most important problems that affect food
freshness, and with it food quality, are exposure time in
an inadequate environment, incorrect design of the food
packaging, inadequate management of temperatures

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CHILEAN J. AGRIC. RES. - VOL. 69 - N 2 - 2009

Table 1. Most important biosensors used in the detection of pesticides, fertilizers and other pollutants.

Analyte

Type of
interaction

Recognition
biocatalyzer

Transduction
system

References

Pesticides
Parathion
Biocatalytic Parathion hydrolase
Amperometric


Propoxur and carbaryl Biocatalytic Acetyl cholinesterase
Fiber optic

Diazinon and dichlorvos Biocatalytic Tyrosinase
Amperometric

Paraoxon
Biocatalytic Alkaline phosphatase
Optical

Velasco-Garca y
Mottram, 2003;
Parellada et al., 1998
Nunes et al., 1998;
Xavier et al., 2000
Prez Pita et al., 1997;
Mello y Kubota, 2002
Cosnier et al., 1998;
Mello and Kubota,
2002; Patel, 2002

Fertilizers
Nitrate
Biocatalytic
Nitrite
Biocatalytic
Phosphate
Biocatalytic

Nitrate reductase
Nitrite reductase
Polyphenol oxidase and
alkaline phosphatase,
phosphorylase A,
phosphoglucomutase and
glucose-6-phosphate
dehydrogenase

Amperometric
Optical
Amperometric

Moretto et al., 1998

Moretto et al., 1998
Cosnier et al., 1998

Heavy metals
Copper and mercury
Biocatalytic Spirulina subsalsa
Amperometric

Copper
Biocatalytic Recombinant Saccharomyces Amperometric

cerevisiae
Cadmium and lead
Biocatalytic Staphylococcus aureus or
Optical

Recombinant Bacillus subtilis
Arsenic, cadmium and Biocatalytic Cholinesterase
Electrochemical
bismuth
Cadmium, copper,
Biocatalytic Ureasa
Optical
chrome, nickel, zinc
Copper and mercury
Biocatalytic Glucose oxidase
Amperometric

and the level of oxygen during the handling of fruit and

vegetables in modified atmospheres, among many others.
Because of this, experimental use has been made of
commercial biosensors that use immobilized enzymes like
alcohol oxidase and alcohol peroxidase and a chromogene,
in which alcohol oxidase catalyzes the oxidation of ethanol
in acetaldehyde and H2O2 in the presence of O2, and the
peroxidase catalyzes the oxidation of the chromogene,
causing a change in color. Smyth et al. (1999), measuring
with biosensors, ethanol accumulation in lettuce (Lactuca
sativa L.), cauliflower (Brassica oleracea var. botrytis),

Tsai, 2003; VelascoGarca and Mottram, 2003

Tsai, 2003; VelascoGarca and Mottram, 2003
Tsai, 2003; VelascoGarca and Mottram, 2003
Tsai, 2003; VelascoGarca and Mottram, 2003
Tsai, 2003; VelascoGarca and Mottram, 2003
Tsai, 2003; VelascoGarca and Mottram, 2003

broccoli (Brassica oleracea var. italica) and cabbage

(Brassica oleracea var. capitata) lightly processed and
packed in a modified atmosphere, detected lesions due to
low concentration of O2 and obtained a response from the
biosensor that was very similar to that obtained by gas
chromatography, which is costly and requires technical
experts. This biosensor can also be used monitor ethanol
formation during apple storage in a controlled atmosphere,
the development of putrefaction in tubercles like potatoes
or for any other application where ethanol accumulation
can be associated with quality loss. Likewise, research has

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L. SERNA et al. - USE OF ENZYMATIC BIOSENSORS AS QUALITY INDICES

Table 2. Some of the most commonly used biosensors in antinutrient detection.

Analyte

Type of
interaction

Recognition
system

Transduction
system

References

Antinutrients
Oxalate (spinaches, tea, strawberries) Biocatalytic Oxalate oxidase
Amygdalin (bitter almonds)
Biocatalytic -glucosidase

Glucoalcaloides
Biocatalytic Cholinesterase
been conducted that analyzes the content of some organic
acids and sugars as indicators of fruit and vegetable
maturity (ngeles and Caizares, 2004).
There are multiple compounds that give rise to
disagreeable flavors and aromas that can be detected
with biosensors, as in the case of 2,4,6-tricloroanisole
in wine (Moore et al., 2003), which is related to wine
bottle corks, whose presence causes significant losses to
the wine industry. In other cases, the level of freshness
of fish has been detected through a hydrogen peroxide
electrode based on the xanthine oxidase enzyme (Volpe
and Mascini, 1996). Biosensors can also detect indicators
of processes, such as lactulose, disaccharide, which
is formed in the thermal treatment of milk allows for
distinguishing between milk that has been submitted to a
UHT treatment (ultra high temperature) and milk sterilized
in the container.
Applications in process control. Currently, thanks
to biosensor technology it is possible to determine and
quantify on-line diverse compounds of importance in
process control, such as sugars, alcohols, and amino acids,
among others.
Sugars are limiting factors in fermentative processes
given that low concentrations reduce the productivity of
the bioreactor. Because of this, numerous investigations
have been undertaken, among which notably are those
on the use of amperometric biosensors to analyze
glucose with glucose oxidase in fruit juices (ngeles
and Caizares, 2004); lactose with -galactosidase and
glucose oxidase; and lactulose with -galactosidase and
fructose dehydrogenase, which implies an excessive
thermal treatment of milk during pasteurization (Camps
et al., 2002). In relation to alcohols and principally
ethanol, enzymatic reactions are inhibited when alcohol
content exceeds 14%; analysis has been advanced mainly
with the alcohol dehydrogenase enzyme Gluconobacter
oxydans with amperometric biosensors; likewise, with
fermentation, the proportion of glycerol should be
maintained at 1:10 in relation to total alcohol, the analysis
of glycerol has been developed with glycerokinase
and glycerol-3-phosphate oxidase in amperometric
biosensors to monitor fermentative processes (Niculescu
et al., 2003). On the other hand, aminoacids like lysine,

Amperometric
Amperometric
Potentiometric
Potentiometric

Milardovic et al., 2000

Ohashi and Karube, 1993
Ohashi and Karube, 1993

obtained by fermentation and employed as animal feed

supplements, has been controlled by the lysine oxidase
enzyme. Similarly, lactic acid used to control acidity and
the formation of crusts on cheese, have been examined
with lactate oxidase in amperometric biosensors. These
biosensors can be integrated into the system of Hazard
Analysis and Critical Control Points (HACCP) to verify
that processes are being carried out correctly.
Despite the broad applicability of biosensors, the use
of the technology in the control of processes is limited for
several reasons: the short life of enzymatic biosensors, the
need to calibrate them with certain frequency, the lack of
reliable response to different concentrations or with variable
conditions in the medium, among others (Ferreira et al.,
2003). The prototype tests in real samples have critical
stages such as immobilization of the biocomponent during
the construction of the device and preparation of the sample
for analysis. Biosensors require mild temperature and pH
conditions to keep the biological element active (Gibson,
1999; Wilson and Gifford, 2005). Consequently, in some
cases a previous treatment of the sample is recommended
to eliminate interfering species such as ascorbic acid,
tyrosine and others. Procedures are conducted that
include neutralization, dilution or extraction when the
food is acidic or hydrophobic. The correction methods
to reduce the duration of food analysis include acidic or
alkaline hydrolysis, microwave digestion, extraction of
supercritical fluids, evaporation and filtration (Deng and
Dong, 1996; Marconi et al., 1996; Kotsira and Clonis,
1998; Panfili et al., 2000). Likewise, specific sensors have
been developed for the determination of glucose, lactate,
glutamate, pyruvate, choline and acetylcholine through
monitoring of nitric oxide, Na+, K+, Ca2+, and dopamine
(Zhang and Wilson, 1998; Wilson and Gifford, 2005).
CONCLUSIONS
The food industry is benefitting from major advances
in the development of enzymatic biosensors with different
transduction systems that can be applied in the areas of
food safety, quality and process control; studies are focused
mainly on determining composition, contamination of
primary materials and processed foods.

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CHILEAN J. AGRIC. RES. - VOL. 69 - N 2 - 2009

Table 3. Most important biosensors applied to evaluate food quality.

Analyte

Matrix

Glucose

Grape juice, wine, juice,

honey, milk and yogurt

Fructose

Juice, honey, milk, gelatin

and artificial edulcorants

Lactose

Milk

Lactate

Cider and wine

Lactulose

Milk

L-amino
acids
L-glutamate

Milk and fruit juices

L-lysine
L-malate

Soya sauce and

condiments
Milk, pasta and
fermentation samples
Wine, cider and juices

Ethanol

Beer, wine and other

alcoholic drinks

Glycerol

Wine

Catechol
Cholesterol

Beer
Butter, lard and egg

Citric acid
Lecithin

Juice and athletic drinks

Egg yolk, flour and soya
sauce

Recognition
enzyme
Glucose oxidase

Transduction
system
Amperometric

Fructose dehydrogenase,
D-fructose 5dehydrogenase
-Galactosidase

Amperometric

Bassi et al., 1998;

Palmisano et al., 2000

Amperometric

Transaminase and Llactate dehydrogenase

Fructose dehydrogenase
and -galactosidase
D-amino acid oxidase

Amperometric
Amperometric

Marconi, 1996; Palmisano

et al., 2000
Silber et al., 1994;
Ramanathan et al., 2001
Sekine and Hall, 1998

Amperometric

Sarkar et al., 1999

L-glutamate oxidase

Amperometric

Lysine oxidase

Amperometric

Dehydrogenated malate,
others
Alcohol oxidase, alcohol
dehydrogenase, NaDH
oxidase
Glycerophosphate
oxidase and glycerol
kinase
Polyphenol oxidase
Cholesterol oxidase and
peroxidase
Citrate lyase
Phospholipase D and
choline oxidase

Amperometric

Matsumoto et al., 1998;

Kwong et al., 2000
Kelly et al., 2000;
Olschewski et al, 2000
Miertus et al., 1998

In the area of food safety, enzymatic biosensors allow for

identifying the presence of highly toxic organic contaminants
and the presence of anti-nutritional elements that affect the
food chain, either accidently or by intention. This early
detection protects the environment from contaminants and
consumers from chronic illnesses and allergies.
Equally, enzymatic biosensors are being used in the
food industry to determine the freshness of products given
that it is possible to detect enzymes and compounds of
aroma and flavor that originate from the senescence stage
of products.
Biosensors have proven to be especially useful in
the control of fermentative processes in follow-up of the
consumption of the substrate by microorganisms, control
of acidity and assessing the thermal profile.

References
Centonze et al., 1997;
ngeles y Caizares, 2004

Amperometric

Katrlk, 1998 ; Miertus et

al., 1998

Amperometric

Niculescua et al., 2003

Amperometric
Amperometric

Eggins et al., 1997

Akyilmaz and Dinckaya,
2000.
Prodromidis et al., 1997
Mello and Kubota, 2002

Amperometric
Electrochemical

While the use of biosensors in the food industry is on

a mass scale, there are still obstacles to be overcome, such
as the high cost of purifying the enzymes that are used as
detecting elements, the low specificity and low response
time that are obtained when complete cells or tissue are
used, the lack of reliable responses low concentrations,
interference reactions, the need to calibrate the devices
and the stability of the enzymes. This last factor is the
most limiting for the lifetime of enzymatic biosensors. If
these limiting factors can be overcome, it will be possible
to develop enzymatic biosensors that are more rapid,
versatile, reliable, long lasting and cost-effective.

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L. SERNA et al. - USE OF ENZYMATIC BIOSENSORS AS QUALITY INDICES

Cuadro 4. Biosensor used in the evaluation of freshness and self life.

Analyte

Matrix

Recognition
enzyme

Transduction
system

References

Evaluation of spoilage
Polyphenols

Olive oil

Tyrosinase, laccase

Amperometric

Short chain
fatty acid

Milk and derivatives

Lipase

Electrochemical

Campanella et al., 1993;

Kulys and Vidziunaite, 2003
Mello and Kubota, 2002

Ornititne carbamoyl
transferase, nucleoside
phosphorylase and
xanthine oxidase
Diamine oxidase,
Ornithine carbamoyl
transferase, nucleoside
phosphorylase
Amine oxidase and
peroxidase
Xanthine oxidase

Amperometric

Mello and Kubota, 2002

Amperometric

Park et al., 2000; Mello and

Kubota, 2002

Amperometric

Tombelli and Mascini, 1998

Amperometric

Hu et al., 2000

Xanthine oxidase,
diamine oxidase
Polymide oxidase

Amperometric

Mello and Kubota, 2002

Electrochemical
Electrochemical

Ramanathan et al., 2001

Mello and Kubota, 2002

Potentiometric

Mello & Kubota, 2002

Freshness index
Ornithine
and amines

Shrimps

Amines

Fish, lobster

Biogenic
amines
Hypoxan
thine
Lactic acid

Fish
Fish
Meat

Evaluation of maturity
Glucose
Sucrose

Fruit
Fruit

Isocitrate

Fruit

Glucose oxidase
Invertase, mutarotase and
glucose oxidase
Isocitrate dehydrogenase

RESUMEN
Uso de biosensores enzimticos como indicadores
de calidad: Una sinopsis del presente y futuro en la
industria alimentaria. Los biosensores constituyen
una importante alternativa en la industria de alimentos
para garantizar la calidad e inocuidad de los productos
y controlar los procesos con mtodos eficaces, rpidos y
econmicos; su tecnologa est basada en un elemento de
reconocimiento biolgico especfico en combinacin con
un transductor para el procesamiento de la seal. El uso de
tcnicas de biosensores enzimticos en procesamiento de
alimentos, control de calidad y de procesos on line, es
prometedor frente a las tcnicas analticas convencionales,
ya que ofrecen grandes ventajas debido a su tamao, costo,

especificidad, respuesta rpida, precisin y sensibilidad.

En este artculo se revisa el desarrollo y uso de algunos
biosensores enzimticos en la industria alimentaria,
se describen las reas de aplicacin ms importantes
y se analiza su situacin actual y posibilidades futuras.
En conclusin, los biosensores enzimticos son una
herramienta de gran aplicabilidad en el desarrollo de
sistemas de calidad como el anlisis de riesgos y puntos
crticos de control, y que la masificacin de su uso en la
industria alimentaria se ve an limitada principalmente
por el tiempo de vida til de los biosensores, para lo cual
se propone el uso de enzimas termoflicas.
Palabras clave: biosensores, enzimas, anlisis en
alimentos, seguridad, calidad, control de procesos.

278

CHILEAN J. AGRIC. RES. - VOL. 69 - N 2 - 2009

LITERATURE CITED
Akyilmaz, E., and E. Dinckaya. 2000. A mushroom
(Agaricus bisporus) tissue homogenate based alcohol
oxidase electrode for alcohol determination in serum.
Talanta 53:505-509.
ngeles A., y M. Caizares. 2004. Desarrollo de un
sistema sensor para la cuantificacin de glucosa en
jugos de frutas. Rev. Soc. Qum. Mx. 8:106-110.
Arroyo, M. 1998. Inmovilizacin de enzimas. Fundamentos,
mtodos y aplicaciones. Ars Pharmaceutica 39:23-39.
Bachmann, T.T., B. Leca, F. Vilatte, J.L. Marty, D.
Fournier, and R.D. Schmid. 2000. Improved
multianalyte
detection
of
organophosphates
and carbamates with disposable multielectrode
biosensors using recombinant mutants of Drosophila
acetylcholinesterase and artificial neural networks.
Biosens. Bioelectron. 15:193-201.
Bassi, A.S., E. Lee, and J.X. Zhu. 1998. Carbon paste
mediated, amperometric, thin film biosensors
for fructose monitoring in honey. Food Research
Internacional 31:119-127.
Campanella, L., T. Beone, M.P. Sammartino, and M.
Tomassetti. 1993. Analysis of L-dopa in pharmaceutical preparations and of total phenols content in
urine by means of an enzyme-amperometric sensor. J.
Pharm. Biomed. Anal. 11:1099-1104.
Camps, M., M. Mir, C. OSullivan, y I. Katakis. 2002.
Biosensores al servicio de la industria alimentaria.
In 2 Congreso Espaol de Ingeniera de Alimentos,
Lleida, Espaa.
Centonze, D., C.G. Zambonin, and F. Palmisano. 1997.
Determination of glucose in nonalcoholic beverages by
a biosensor coupled with microdialysis fiber samplers.
Journal of AOAC International 80:829-833.
Cosnier, S., C. Gondran, J.C. Watelet, W. De Giovani,
R.P.M. Furriel, and F.A. Leone. 1998. A bienzyme
electrode (alkaline phosphatase-polyphenol oxidase)
for the amperometric determination of phosphate.
Anal. Chem. 70:3952-3956.
Davis, J., D. Huw Vaughan, and M.F. Cardosi. 1995.
Elements of biosensors construction. Enzyme Microb.
Technol. 17:1030-1035.
Deng, Q., and S. Dong. 1996. Amperometric biosensor for
tyrosinase inhibitors in a pure organic phase. Analyst
121:1979-1982.
DOrazio, P. 2003. Biosensors in clinical chemistry. Clin.
Chim. Acta 334:41-69.
DSouza, S.F. 2001. Microbial biosensors. Biosen.
Bioelectron. 16:337-353.
Eggins, B.R., C. Hickey, S.A. Toft, and D.M. Zhou.
1997. Determination of flavonols in beers with tissue
biosensors. Anal. Chim. Acta 347:281-288.

Ferreira, S., M.B. De Souza, J.O. Trierweiler, O.

Broxtermann, R.O.M. Folly, and B. Hitzmann. 2003.
Aspects concerning the use of biosensors for process
control: experimental and simulation investigations.
Comput. Chem. Eng. 27:1165-1173.
Fitzpatrick, J., L. Fanning, S. Hearty, P. Leonard, B.M.
Manning, J.G. Quinn, and R. OKennedy. 2000.
Applications and recent developments in the use of
antibodies for analysis. Anal. Lett. 33:2563-2609.
FSA. 2004. An investigation of the use of terms such as
natural, fresh etc. in food labeling. Survey report of
the February 11, 2004, Food Labelling and Standards
Division, Food Standards Agency. (FSA), UK.
Gajovic, N., G. Binyamin, A. Warsinke, F.W. Scheller,
and A. Heller. 2000. Operation of a miniature
redox hydrogel-based pyruvate sensor in undiluted
deoxygenated calf serum. Anal. Chem. 72:29632968.
Gibson, T.D. 1999. Biosensors: the stability problem.
Analusis 27:630-638.
Glazier, S.A., M.A. Arnold, and J.P. Glazier. 1988.
Evaluation of an overdetermined system based on
multiple ion-selective electrodes of the same type.
Talanta 35:215-219.
Hall, R.H. 2002. Biosensor technologies for detecting
microbiological foodborne hazards. Microbes and
Infect. 4:425-432.
Hedenmo, M., A. Narvez, E. Domnguez, and I. Katakis.
1997. Improved mediated tyrosinase amperometric
enzyme electrodes. J. Electroanal. Chem. 425:1-11.
Hu, S., C. Xu, J. Luo, J. Luo, and D. Cui. 2000. Biosensor
for detection of hypoxanthine based on xanthine
oxidase immobilized on chemically modified carbon
paste electrode. Anal. Chim. Acta 412:55-61.
Katrlk, J., J. Svorc, M. Stredansky, and S. Miertus. 1998.
Composite alcohol biosensors based on solid binding
matrix. Biosens. Bioelectron. 13:183-191.
Kelly, S.C., P.J. OConnell, C.K. OSullivan, and G.G.
Guilbault. 2000. Development of an interferent free
amperometric biosensor for determination of L-lysine
in food. Anal. Chim. Acta 412:111-119.
Killard, A.J., and M.R. Smyth. 2000. Separation-free
electrochemical immunosensor strategies. Anal. Lett.
33:1451-1465.
Kotsira, V.P., and Y.D. Clonis. 1998. Colorimetric assay
for lecithin using two co-immobilized enzymes and
an indicator dye conjugate. J. Agric. Food Chem.
46:3389-3394.
Krajewska, B. 2004. Application of chitin- and chitosanbased materials for enzyme immobilizations: a review.
J. Biotechnol. 35:126-139.

L. SERNA et al. - USE OF ENZYMATIC BIOSENSORS AS QUALITY INDICES

Kwong, A.W.K., B. Grndig, J. Hu, and R. Renneberg.

2000. Comparative study of hydrogel-immobilized
L-glutamate oxidases for a novel thick-film biosensor
and its application in food samples. Biotechnol. Lett.
22:267-272.
Kulys, J., and R. Vidziunaite. 2003. Amperometric
biosensors based on recombinant laccases for phenols
determination. Biosen. and Bioelectron. 18:319-325.
Laschi, S., M. Frnek, and M. Mascini. 2000. Screenprinted electrochemical immunosensors for PCB
detection. Electroanalysis 12:1293-1298.
Li, B., Z. Zhang, and Y. Jin. 2002. Plant tissuebased chemiluminescence flow biosensor for
determination of unbound dopamine in rabbit blood
with on-line microdialysis sampling. Biosens. and
Bioelectron.17:585-589.
Luong, J.H.T, C.A. Groom, and K.B. Male. 1991. The
potential role of biosensors in the food and drink
industries. Biosens. and Bioelectron. 6:547-554.
Luong, J.H.T., K.B. Male, and J.D. Glennon. 2008.
Biosensor technology: technology push versus market
pull. Biotechnol. Adv. 26:492-500.
Marconi, E., G. Panfili, M.C. Messia, R. Cubadda, D.
Compagnone, and G. Palleschi. 1996. Fast analysis of
lysine in food using protein microwave hydrolysis and
an electrochemical biosensor. Anal. Lett. 29:1125-1137.
Matsumoto, K., W. Asada, and R. Murai. 1998.
Simultaneous biosensing of inosine monophosphate
and glutamate by use of immobilized enzyme reactors.
Anal. Chim. Acta 358:127-136.
Mei, Y., L. Ran, X. Ying, Z. Yuan, and S. Xin. 2007. A
sequential injection analysis/chemiluminescent plant
tissue-based biosensor system for the determination of
diamine. Biosens. Bioelectron. 22:871-876.
Mello, L.D., and L.T. Kubota. 2002. Review of the use
of biosensors as analytical tools in the food and drink
industries. Food Chem. 77:237-256.
Miertus, S., J. Katrlk, A. Pizzariello, M. Stredansk, J.
Svitel, and J. Svorc. 1998. Amperometric biosensors
based on solid binding matrices applied in food quality
monitoring. Biosens. Bioelectron. 13:911-923.
Milardovic, S., Z. Grabaric, and B.S. Grabaric. 2000.
Sensitive amperometric oxalate biosensor for food
analysis. Food Technol. Biotechnol. 38:203-210.
Moore, E., M. Pravda, and G.G. Guilbault. 2003.
Development of a biosensor for the quantitative
detection of 2,4,6-trichloroanisole using screen printed
electrodes. Anal. Chim. Acta 484:15-24.
Morales, M.D., S. Morante, A. Escarpa, M.C. Gonzlez,
A.J. Reviejo, and J.M. Pingarrn. 2002. Design of a
composite amperometric enzyme electrode for the
control of the benzoic acid content in food. Talanta
57:1189-1198.

279

Moretto, L.M., P. Ugo, M. Zanata, P. Guerriero, and C.R.

Martin. 1998. Nitrate biosensor based on the ultrathinfilm composite membrane concept. Anal. Chem.
70:2163-2166.
Niculescu, M., R. Mieliauskiene, V. Laurinavicius, and
E. Csregi. 2003. Simultaneous detection of ethanol,
glucose and glycerol in wines using pyrroloquinoline
quinone dependent dehydrogenases based biosensors.
Food Chem. 82:481-489.
Nunes, G.S., P. Skladal, H. Yamanaka, and D. Barcelo. 1998.
Determination of carbamate residues in crop samples by
cholinesterase-based biosensors and chromatographic
techniques. Anal. Chim. Acta 362:59-68.
Odaci, D., S. Timur, and A. Telefoncu. 2004. Carboxyl
esterase-alcohol oxidase based biosensor for the
aspartame determination. Food Chem. 84:493-496.
Ohashi, E., and I. Karube. 1993. Sensors for the food
industry. Food Control 4:183-188.
Olschewski, H., A. Erlenktter, C. Zaborosch, and G.C.
Chemnitius. 2000. Screen-printed enzyme sensors
for l-lysine determination. Enzyme Microb. Technol.
26:537-543.
Palmisano, F., R. Rizzi, D. Centonze, and P.G. Zambonin.
2000. Simultaneous monitoring of glucose and lactate
by an interference and cross-talk free dual electrode
amperometric biosensor based on electropolymerized
thin films. Biosens. Bioelectron. 15:531-539.
Panfili, G., P. Manzi, D. Compagnone, L. Scarciglia, and
G. Palleschi. 2000. Rapid assay of choline in foods
using a microwave hydrolysis and a choline biosensor.
J. Agr. Food Chem. 48:3403-3407.
Parellada, J., A. Narvez, M.A. Lpez, E. Domnguez,
J.J. Fernndez, V. Pavlov, and I. Katakis. 1998.
Amperometric immunosensors and enzyme electrodes
for environmental applications. Anal. Chim. Acta
362:47-57.
Park, I.-S., Y.-J. Cho, and N. Kim. 2000. Characterization
and meat freshness application of a serial threeenzyme reactor system measuring ATP-degradative
compounds. Anal. Chim. Acta 404:75-81.
Patel, P.D. 2002. (Bio)sensors for measurement of
analytes implicated in food safety: a review. Trends
Anal. Chem. 21:96-115.
Prez Pita, M.T., A.J. Reviejo, F.J. Manuel de Villena,
and J.M. Pingarrn. 1997. Amperometric selective
biosensing of dimethyl- and diethyldithiocarbamates
based on inhibition processes in a medium of reversed
micelles. Anal. Chim. Acta 340:89-97.
Prodromidis,
M.I.,
S.M.
Tzouwara-Karayanni,
M.I. Karayannis, and P.M. Vadgama. 1997.
Bioelectrochemical determination of citric acid in
real samples using a fully automated flow injection
manifold. Analyst 122:1101-1106.

280

Ramanathan, K., B.R. Jnsson, and B. Danielsson. 2001.

Sol-gel based thermal biosensor for glucose. Anal.
Chim. Acta 427:1-10.
Rasooly, A. 2001. Surface plasmon resonance analysis of
Staphylococcal enterotoxin B in food. Food Protec.
64:37-43.
Rogers, K.R., and M. Mascini. 1998. Biosensors for field
analytical monitoring. Field Anal. Chem. Technol.
2:317-331.
Saidman, S.B., M.J. Lobo-Castan, A.J. MirandaOrdieres, and P. Tun-Blanco. 2000. Amperometric
detection of D-sorbitol with NAD+-D-sorbitol
dehydrogenasse modified carbon paste electrode.
Anal. Chim. Acta 424: 45-50.
Sarkar, P., I.E. Tothill, S.J. Setford, and A.P.F. Turner.
1999. Screen-printed amperometric biosensors for the
rapid measurement of L- and D-amino acids. Analyst
124:865-870.
Sekine, Y., and E.A.H. Hall. 1998. A lactulose sensor
based on coupled enzyme reactions with a ring
electrode fabricated from tetrathiafulvalen-tetracyanoquinodimetane. Biosens. Bioelectron. 13:995-1005.
Setti, L., A. Fraleoni-Morgera, I. Mencarelli, A. Filippini,
B. Ballarin, and M. Di Biase. 2007. An HRP-based
amperometric biosensor fabricated by thermal inkjet
printing. Sensor Actuator B- Chem. 126:252-257.
Silber, A., C. Bruchle, and N. Hampp. 1994.
Dehydrogenase-based thick-film biosensors for lactate
and malate. Sensor Actuator B- Chem. 18:235-239.
Smyth, A.B., P.C. Talasila, and A.C. Cameron. 1999. An
ethanol biosensor can detect low-oxygen injury in
modified atmosphere packages of fresh-cut produce.
Postharvest Biol. Technol. 5:127-134
Tatsumi, H., H. Katano, and T. Ikeda. 2006. Kinetic
analysis of enzymatic hydrolysis of crystalline
cellulose by cellobiohydrolase using an amperometric
biosensor. Anal. Biochem.357:257-261.

CHILEAN J. AGRIC. RES. - VOL. 69 - N 2 - 2009

Tingry, S., C. Innocent, S. Touil, A. Deratani, and P. Seta.

2006. Carbon paste biosensor for phenol detection
of impregnated tissue: modification of selectivity
by using -cyclodextrin-containing PVA membrane.
Mater. Sci. Eng. C 26:222-226.
Tombelli, S., and M. Mascini. 1998. Electrochemical
biosensors for biogenic amines: A comparison between
different approaches. Anal. Chim. Acta 358:277-284.
Tothill, I.E. 2001. Biosensors developments and potential
applications in the agricultural diagnosis sector. Comp.
Electron. Agr. 30:205-218.
Tsai, H.C., R.A. Doong, H.C. Chiang and K.T. Chen.
2003. Sol-gel derived urease-based optical biosensor
for the rapid determination of heavy metals. Anal.
Chim. Acta 481:75-84.
Vadiuambal, R., and D.S. Jayas. 2007. Changes in quality
of microwave-treated agricultural products: a review.
Biosyst. Eng. 98:1-16.
Velasco-Garca, M.N., and T. Mottram. 2003. Biosensor
technology addressing agricultural problems. Review
paper. Biosyst. Eng. 84:1-12.
Volpe, G., and M. Mascini. 1996. Enzyme sensors for
determination of fish freshness. Talanta 43:283-289
Wilson, G.S., and R. Gifford. 2005. Review biosensors for
real-time in vivo measurements. Biosens. Bioelectron.
20:2388-2403.
Xavier, M.P., B. Vallejo, M.D. Marazuela, M.C. MorenoBondi, F. Baldini, and A. Falai. 2000. Fiber optic
monitoring of carbamate pesticides using porous glass
with covalently bound chlorophenol red. Biosens.
Bioelectron. 14:895-905.
Zhang, Z., and R.P. Wilson. 1998. A modified enzymatic
assay for quantifying choline in fish tissue and common
feed ingredients. J. Agr. Food Chem. 46:3673-3676.
Zinedine, A., J.M. Soriano, J.C Molto, and J. Maes.
2007. Review on the toxicity, occurrence, metabolism,
detoxification, regulations and intake of zearalenone: an
oestrogenic mycotoxin. Food Chem. Toxicol. 45:1-18.